Miltenyi VYB SOP

VYB SOP (March, 2018)

1. Check that the sheath fluid tank is filled (if not pour running buffer found in the closet behind you into the tank) and the waste tank is half empty (if not pour the content into the carboy located in the sink + add bleach to 10% final.

2. Touch the screen to initialize the software (~5 min). The instrument is now is analysis mode

3. Login

4. Switch to acquisition mode by clicking on the icon located on the top right  

5. Run Calibration:

·       Take the tube rack and connect it

·       Place the bottle of beads from fridge on the rack to scan the bar code, click on the bar code icon

·       Follow instruction by placing a FACS tube on the tube rack, adding one drop of beads in the tube and clicking OK

·       If calibration does not pass, do a clean/flush cycle and email Marielle and Nandhini with the issue

6. Open your Analysis Template (private folder). Default templates can be found in the public folder. To modify the parameters, click on A tab, which will allow you to modify gates and change parameters.             

7. Open your Instrument Settings (private folder). Default templates can be found in the public folder. Choose the one with the same type of cells.

8. In experiment Tab, select 96 well plate or single tube (for plates: verify that the block and the plate are positioned). Run “Setup Samples” to make sure the setting and OK; Adjust voltages if needed

9. If set up is OK, Acquire your tubes one by one making sure that the setting are OK (number of event, stopping gate, volume of samples, volume of the samples to be run, speed, washes…)

For tubes:

·       When running tubes, the file name will be “initial_date_001”. To also record a more descriptive name use the box “sample write the name of your sample in the “sample ID” box. This “sample ID” can be displayed in flowjo as column $CELLS (see below)

For plates:

·       Open/Experiment/Public and chose Plate1 in order to import the names of the wells (Plate1A1, Plate1A2…) for that plate. To delete the wells that are empty, select with red ring.  Right click on the wells that have no sample, and click on “clear selected” in the pop up menu). All wells with samples to be run should be in gray. All empty wells should be clear.

·       Select the all wells (ie have orange ring) and verify that the parameters are correct going from bottom to top (number of event, stopping gate, volume of samples, volume of the samples to be run, speed, washes…). With all the wells to be run selected.

·       Run plate (play button low right).

10. To transfer the data to your computer,

·       Connect the VYB to the network

·       Close the template by clicking on A and close template tab

·       Click on the CD icon (top bar) and back up data

11. Log out.

Clogging issues => The drop icon on the bottom right gives you the wash/clean/flush cycles (first thing to do if problems occur: Right click and choose clean (7min cycle)+ right click and choose flush (15min cycles). Otherwise contact Flow Core Staff or online support (Tool tab)

Exporting FCS files => To export FCS files, go to Edit, option, software, acquire and click FCS format, click apply & OK

FlowJo analysis=>To retrieve the “sampleID”, in the flowjo workspace, right click on the column of the workspace, select $CELLS in the column values, add column, and click ok